Pseudomycins useful against plant diseases

ABSTRACT

Plants and crops subject to attack by fungal related diseases are protected or treated by the application of  Pseudomycin  compositions which were originally isolated from  Pseudomonas syringae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application US01/25724, filedAug. 17, 2001, and to U.S. Provisional Application Ser. No. 60/226,010,filed Aug. 18, 2000.

FIELD OF THE INVENTION

The present invention relates to the use of pseudomycins as effectivefungicides against plant and crop diseases, and more particularlyrelates to the use of pseudomycins against particular classes of fungiwhich cause diseases in plants and crops.

BACKGROUND

One class of new antifungal agents, the pseudomycins, shows greatpromise for treating fungal infections in a variety of patients. (seei.e., Harrison, L., et al., “Pseudomycins, a family of novel peptidesfrom Pseudomonas syringae possessing broad-spectrum antifungalactivity,” J. Gen. Microbiology 137(12), 2857-65 (1991) and U.S. Pat.Nos. 5,576,298 and 5,837,685). Pseudomycins are natural products derivedfrom isolates of Pseudomonas syringae. P. syringae is a large group ofplant-associated bacteria that have been the source of several bioactivesubstances, such as bacitracin and the syringomycins. Natural strainsand transposon-generated mutants of P. syringae produce compounds withantifungal activity. A transposon-generated regulatory mutant of thewild type strain of P. syringae MSU 174, known as MSU 16H (ATCC 67028),produces several pseudomycins. Pseudomycins A, B, C and C′ have beenisolated, chemically characterized, and shown to possess wide spectrumantifungal activity, including activity against important fungalpathogens in both humans and plants. The pseudomycins are structurallyrelated to but are distinct from syringomycin and other antimycoticsfrom isolates of P. syringae. The peptide moiety for pseudomycins A, B,C, C′ corresponds toL-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) withthe terminal carboxyl group closing a macrocyclic ring on the OH groupof the N-terminal Ser. The analogs are distinguished by the N-acyl sidechain, i.e., pseudomycin A is N-acylated by 3,4-dihydroxytetradeconoate,pseudomycin B by 3-hydroxytetradecanoate, pseudomycin C by3,4-dihydroxyhexadecanoate and pseudomycin C′ by 3-hydroxyhexadecanoate.(see i.e., Ballio, A., et al., “Novel bioactive lipodepsipeptides fromPseudomonas syringae: the pseudomycins,” FEBS Letters, 355(1), 96-100,(1994) and Coiro, V. M., et al., “Solution conformation of thePseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin Adetermined by computer simulations using distance geometry and moleculardynamics from NMR data,” Eur. J. Biochem., 257(2), 449-456 (1998).)

The present invention provides a group of pseudomycins which areparticularly useful to protect plants and crops against fungal diseases.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for thetreatment or protection of plants and crops against diseases.

A further object of the invention is to provide a method for thetreatment or protection of plants and crops by application of certainpseudomycins.

An even further object of the invention is the use of certainpseudomycins to protect or treat plants and crops against diseasescaused by fungi.

Other objects and advantages of the invention will become apparent asthe description of the invention proceeds.

In satisfaction of the foregoing objects and advantages, the presentinvention provides a method for the protection or treatment of plant andcrops against fungal-related diseases, which comprises applying to saidplants or crops an effective amount of one or more pseudomycin products.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure relates to the discovery of novel, previouslyunsuspected uses for the class of lipopeptides known collectively as thepseudomycins as fungicides or antimycotic agents. In a preferredembodiment, the pseudomycins, are individually and as a group,particularly useful in the treatment or protection of plants challengedby a group of Ascomyceteous fungi related to Mycosphaerella asp.(perfector sexual stage of the fungus) and virtually all of the variousimperfect stages of this fungus that are known, including Septoria sp.,and Cercospora sp. In addition, a number of other extremely economicallyimportant plant pathogenic fungi are killed by the pseudomycinsincluding Tapesia yallundae, Ustilago maydis, Penicillum roqueforti,Monilinia sp. and Geotrichum candidum. Thus, the pseudomycins, alone orindividually, have use in treating plants to protect them from harmcaused by these fungi.

The types of diseases caused by these organisms range from plants instorage (fruits and vegetables) to extremely important field diseasessuch as Black Sigatoka of banana and straw breaker and blotch of wheat.

This discovery relates to a previously unsuspected set of extremelyimportant plant pathogenic fungi that seem to be biologically relatedand are sensitive to one or more of the pseudomycins and are bothinhibited and killed by them. These fungi and a few others, notpreviously disclosed, cause some of the most important plant diseases onthe planet. Currently these fungal-caused diseases are being controlledby simple or more complex mixtures of man-made fungicides which causeenvironmental damage and may be an unsuspected threat to human health.The pseudomycins, on the other hand, offer a safe, and effectivealternative to the use of man-made chemicals for the control of certainplant diseases. In addition, the use of the pseudomycins for plantdisease control have certain benefits since the use of natural productsfor disease control would allow the producer to proclaim that the crophas been grown under biological/organic conditions allowing for a higherprofit to be made on the product. This is noteworthy since no major cropin the world currently has applied to it any naturally producedfungicide for plant disease control. The pseudomycins certainly offermany benefits to both the agricultural producer as well as the consumer.

As an example of how and why the pseudomycins may be useful to theworld's agriculture, the minimum inhibitory concentrations(MICs) forseveral of the pseudomycins e.g. Pseudo A, B, B′C, and C′ are in therange of 1 mg or less per ml. This is an extremely desirableconcentration for effective application in agricultural situations.These compounds produce an even greater effect (less than 1.0 mg) whentested against M. fijiensis isolate 8088/88. M. fijiensis is the causalorganism of the Black Sigatoka disease of bananas and plantains.Currently, the producers of these crops, worldwide, must spray a mixtureof three fungicides (man-made) at the rate of 30 times per year in orderto have a banana crop. This one disease alone represents the largestconsumption of fungicide per crop in the entire world. The disadvantagesfor the use of these synthetic fungicides are numerous including: 1.their extremely high cost ($ millions); 2. the inability of the producerto sell organically grown produce since fungicides have been applied toit; and 3. the uncertainty to human as well the environmental healthrisks involved in the continuous use of the fungicides over decades. Thesoil beneath the banana canopy in the plantations appears sterile ofanimal life and shows a build up of fungicide residues. On the otherhand, the naturally produced pseudomycins appear more effective incontrolling the sigatoka disease, while at the same time offeringbenefits to the environment and to human health.

In addition, the pseudomycins are effective against a number of otherplant disease causing fungi including the fingi that destroy plantproduce in storage e.g. Penicillium sp., Monilinia sp. and Geotrichumsp. A mixture of pseudomycins applied to harvested fruit would precludefungal infections and storage rots.

Still other possibilities for the applications of the pseudomycinsinclude applications for the control of diseases caused by Septoria sp.,specifically S. nodurum and S. triticii on wheat, but also, based on thebiological activity of these molecules-virtually any Septoria sp.causing any plant disease in the world. Likewise, other fungi related toMycosphaeella sp. are affected and they include plant diseases caused byCercospora sp. which causes leaf spot of sugar beets and many othercrops. Other disease causing organisms are also affected by thepseudomycins and they include Dreschslera portulaceae.

In accordance with the present invention it has been discovered that thepseudomycins described herein possess enormous antimycotic activityagainst a previously unsuspected, and closely related group of plantpathogenic fungi. This main group is represented by the perfect stagefungus sp. Mycosphaeella sp. and each of its representatives in theimperfect stage(asexual stage) such as Septoria sp. and Cercospora sp.Generally, the pseudomycins may be used alone or as a mixture in aformulation to protect plants from fungal infection. Applications tocrops in the field as well as in storage are visualized as the potentialuses of these compounds.

Thus, it is a purpose of this invention to demonstrate that a number ofextremely economically important plant pathogenic fungi are susceptibleto the effects of one or more pseudomycins which were originallyisolated from the plant associated bacterium-Pseudomonas syringae.

The pseudomycins useful in the method of the present invention arepreferably pseudomycins produced by the Pseudomonas syringae includingthe pseudomycins identified as Pseudomycins A, A′ B, B′ C and C′ as wellas derivatives such as pseudomycin A-PO4, a phosphate derivative andpseudomycin A-FB, both of which are known.

These pseudomycins are applied against a wide variety of plants andcrops which are susceptible to parasitic diseases caused by fungi. Inthat connection, the pseudomycin compositions of the present inventionare primarily useful to prevent the onset of parasitic diseases causedby fungi so that treatment of the plants and crops prior to onset of thedisease is preferred. However, the pseudomycin compositions are alsouseful in treatment of infected plants.

Pseudomycin compositions of the present invention are effective at verylow concentrations on the order of 1 up to 100 micrograms of pseudomycinper ml of aqueous solution. In that regard, a preferred method ofapplication of the pseudomycin composition of this invention is bytreatment as by spraying directly onto the plant or crop to be treatedusing the indicated concentrations. The pseudomycin compositions of thepresent invention may be in the form of solutions, suspensions oremulsions, or any other form suitable for spraying onto the plants andcrops.

The preferred pseudomycins used in the present invention and theirmethods of preparation are known or are fully disclosed and described incopending applications PCT/US00/08728, filed Apr. 14, 2000 andPCT/US00/08727, filed Apr. 14, 2000 both applications designating theUnited States. The disclosures of both these applications areincorporated herein by reference in their entirety.

As used herein, the term “pseudomycin” refers to compounds having thefollowing formula I:

where R is a lipophilic moiety. The pseudomycin compounds A, A′, B, B′,C, C′ are represented by the formula I above where R is as definedbelow.

Pseudomycin A R = 3,4-dihydroxytetradecanoyl Pseudomycin A′ R =3,4-dihydroxypentadecanoate, Pseudomycin B R = 3-hydroxytetradecanoylPseudomycin B′ R = 3-hydroxydodecanoate Pseudomycin C R =3,4-dihydroxyhexadecanoyl Pseudomycin C′ R = 3-hydroxyhexadecanoyl

As used herein, pseudomycin refers to one or more members of a family ofantifungal agents that has been isolated from the bacterium Pseudomonassyringae. A pseudomycin is a lipodepsipeptide, a cyclic peptideincluding one or more unusual amino acids and having one or moreappended hydrophobic or fatty acid side chains. Specifically, thepseudomycins are lipodepsinonapeptides, with a cyclic peptide portionclosed by a lactone bond and including the unusual amino acids4-chlorothreonine, 3-hydroxyaspartic acid, dehydro-2-aminobutyric acid,and 2,4-diaminobutyric acid. It is believed that these unusual aminoacids are involved in biological characteristics of the pseudomycins,such as stability in serum and their killing action. Pseudomycinsinclude pseudomycin A, pseudomycin A′, pseudomycin B, pseudomycin B′,pseudomycin C, and pseudomycin C′. Each of these pseudomycins has thesame cyclic peptide nucleus, but they differ in the hydrophobic sidechain attached to this nucleus.

Pseudomycins A, A′, B, B′, C and C′ have each been isolated and purifiedand their structures have been characterized by methods including aminoacid sequencing, NMR, and mass spectrometry. Pseudomycins A, B, C, andC′ are discussed in U.S. Pat. No. 5,576,298, issued Nov. 19, 1996 to G.Strobel et al.; Harrison et al., “Pseudomycins, a family of novelpeptides from Pseudomonas syringae possessing broad-spectrum antifungalactivity,” J. Gen. Microbiology 137, 2857-2865 (1991); and Ballio etal., “Novel bioactive lipodepsipeptides from Pseudomonas syringae: thepseudomycins,” FEBS Lett. 355, 96-100 (1994). Pseudomycins A′ and B′ aredescribed in U.S. Pat. Application Ser. No. PCT/US00/08727, byPalaniappan Kulanthaivel, et al. entitled “Pseudomycin Natural Products”submitted even date herewith and exemplified in the Examples, andincorporated herein by reference in its entirety. Antifungal activitydue to several pseudomycins was traced to P. syringae bearing atransposon known as Tn 903, which encodes factors including kanamycinresistance. The sequence of and methods for manipulating transposon Tn903 are known. Oka et al., “Nucleotide sequence of the kanamycinresistance transposon Tn 903,” J. Mol. Biol. 147, 217-226 (1981). Eachof the references cited in this paragraph is specifically incorporatedherein by reference.

The pseudomycins vary in structure and properties. Preferredpseudomycins A, B, C and C′ exhibit activity against a wide variety offungi and also exhibit generally acceptable toxicity. Compared to theother preferred pseudomycins, pseudomycin B has greater potency againstcertain fungi and a lower level of toxicity. Therefore, for the presentmethods, pseudomycin B is more preferred. Each pseudomycin has a cyclicnonapeptide ring having the sequenceSer-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr (Serine; 2,4-Diaminobutyricacid; Aspartic acid; Lysine; 2,4-Diaminobutyric acid; alloThreonine;Dehydro-2-aminobutyric acid; 3-hydroxyAspartic acid; 4-chloroTheonine),more specifically,L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl), withthe carboxyl group of the ClThr and the hydroxyl group of the serineclosing the ring with a lactone bond. The pseudomycins differ in thenature of the lipophilic moiety that is attached to the amine group ofthe N-terminal serine. The amine group of the serine forms an amide bondwith the carboxyl of a 3,4-dihydroxytetradecanoyl moiety in pseudomycinA, a 3-monohydroxytetradecanoyl moiety in pseudomycin B, a3,4-dihydroxyhexadecanoyl moiety in pseudomycin C and a3-monohydroxyhexadecanoyl moiety in pseudomycin C′. The carboxyl groupof the serine forms an amide bond with the Dab of the ring.

The pseudomycins used in the present invention may be used as theiracceptable salts. The term “acceptable salt”, as used herein, refers tosalts of the compounds described above that are substantially non-toxicto living organisms. Typical acceptable salts include those saltsprepared by reaction of the compounds of the present invention with amineral or organic acid or an inorganic base. Such salts are known asacid addition and base addition salts.

Acids commonly employed to form acid addition salts are mineral acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, and phosphoric acid, and organic acids such as p-toluenesulfonic,methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, and acetic acid.Examples of such pharmaceutically acceptable salts are the sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fiunarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, napththalene-2-sulfonate, andmandelate. Preferred pharmaceutically acceptable acid addition salts arethose formed with mineral acids such as hydrochloric acid andhydrobromic acid, and those formed with organic acids such as maleicacid and methanesulfonic acid.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates, andbicarbonates. Such bases useful in preparing the salts of this inventionthus include sodium hydroxide, potassium hydroxide, ammonium hydroxide,potassium carbonate, sodium carbonate, sodium bicarbonate, potassiumbicarbonate, calcium hydroxide, and calcium carbonate. The potassium andsodium salt forms are particularly preferred.

It should be recognized that the particular counterion forming a part ofany salt of this invention is not of a critical nature, so long as thesalt as a whole is pharmacologically acceptable and as long as thecounterion does not contribute undesired qualities to the salt as awhole.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

Biological Materials on Deposit

P. syringae MSU 16H is publicly available from the American Type CultureCollection, Parklawn Drive, Rockville, Md., USA as Accession No. ATCC67028. P. syringae strains 25-B1, 7H9-1, and 67 H1 were deposited withthe American Type Culture Collection on Mar. 23, 2000 and were assignedthe following Accession Nos.:

25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67H1 AccessionNo. PTA-1621

The pseudomycins were isolated from liquid cultures of Pseudomonassyringae. Pseudomonas syringae is a plant-associated microbe producing avariety of phytotoxins and other complex peptides¹⁻³. In the late 1980s,it was shown that P. syringae was producing antifungal agents.Basically, the concept that endosymbionts growing on the plant produceantifungal agents to protect the plant from fungal diseases. Thepseudomycins were identified as the bioactive antifungal agents. Atransposon-generated mutant of P. syringae wild type was shown to behyper-producers of these natural products. These transposon mutantsstrains⁴ developed at Montana State University were used successfully toinoculate elm trees to control Dutch Elm Disease⁵⁻⁶. In addition, thesenatural products have shown selective antifungal activity againstdiseases found on field crops, fruits and other plants (Tables 1-3). Forexample, the pseudomycins shows promising activity against M. fijiensis(M. fijiensis causing black sigatoka of bananas requires more fungicideand fungicide applications than any other plant disease in the worldtoday. The pseudomycins were also shown to prevent premature spoilage ofmangoes.

EXAMPLE 1

Reproducible large-scale production (kilograms) of these naturalproducts has been successfully demonstrated. Purified samples of freebase and alternative salt forms were prepared. Approximately, 34 fungalplant pathogens were used to evaluate the antifungal properties ofpseudomycin A, B, B′, C, C′. Inhibitory concentrations were measured attwo-time points day 2 and 5.

TABLE 1 Antifungal Activity tested with pathogens of interest: a.Alternaria helianthi = leaf spot of sunflower b. Aphanomyces sp. = rootrot, seedling wilt of many plants including sugarbeets c. Bipolarissorokiniana = kernal blight of barley d. Botrytis alli = gray mold neckrot of onion e. Cochliobolis carbonum = leaf blight of corn f. Diplodianatalensis = bunch rot of grapes g. Dreschslera portulacae = leaf spotof portulacs✓ h. D. teres = barley net blotch i. D. tritici-repentisleaf spot of wheat j. Fusarium avenaceum = root rot of several fieldcrops k. F. culmorum = fusarium blight or scab l. F. oxysporum =fusarium vascular wilt m. F. solani = root rot n. Geotrichum candidum =tomato field rot✓ o. Monilinia fructicola = brown rot of stone fruits p.Mycosphaeella fijiensis = black sigatoka of banana✓ (FIG. 3) q.Penicillum roqueforti = green mold of fruit in storage✓ r. Phyllostictamaydis = yellow leaf spot of corn s. Phytophthoras causing blight, rotsdecay t. Rhizoctonia solani = rhizoctonia root rot u. Sclerotinasclerotiorum = crown rot of many plant species v. Septoria tritici =leaf blotch and glume blotch of wheat✓ (FIG. 5) w. Tapesia acuformis =take all disease of wheat x. Ustilago maydis = smut of corn✓ y.Verticillum dahlia = verticillum wilt of many crops and tree species. =Very sensitive towards pseudomycinsMaterials and Methods

Fungi to be tested were propagated on potato dextrose agar (PDA) platesat room temperature. Stock solutions of pseudomycins and depsipeptidewere suspended in dimethylsulfoxide (DMSO) (Sigma) at 5 mg/ml and storedat −20C. Serial dilutions of compounds were done the day of theexperiment. All serial dilutions were made in DMSO. Note: an initialexperiment of pseudomycins B and B′ in methanol was performed on some ofthe fungi, and several fungi showed inhibited growth in the presence ofmethanol. These are noted in the raw data section.

Assays were performed in 24-well cell culture clusters (Costar 3524),with 990 ul potato dextrose broth (PDB, Difco) and 10 ul of the compoundto be tested in each well. Initial concentrations tested were 50-1.56ug/ml (final concentration of pseudomycin). Actual concentrations ofpseudomycins tested were 50, 25, 12.5, 6.25, 3.12 and 1.56 ug/ml. Eachwell was inoculated with the appropriate fungus. Unless noted, thefungal inoculum consisted of an ˜4 mm² piece of PDA with fungalmycelium. The exceptions were as follows. Ustilago maydis: this fungusgrows much like a yeast on PDA, and cells were scraped from the PDAstock plate, resuspended in PDB and 10 ul inoculum added to each well.Monilinia sp.: this fungal mycelium grows as a very loose sheet on topof PDA and would not adhere to agar blocks. Fungal mycelium was groundwith a metal rod in 500 ul PDB and 10 ul inoculum was added to eachwell. Mycosphaeella fijiensis, Septoria passerinii, Septoria triticii:these fungi all grow very slowly and the results were difficult to scoreif a small piece of agar with fungal mycelium on it was used as aninoculum. For these fungi, the mycelium was ground with a metal rod in500 ul PDB and 10 ul inoculum was added to each well.

Wells were scored for fungal growth at two days and five days. Some slowgrowing fungi were scored at later days for growth because assessmentwas not possible at two or five days. Growth was scored by comparison toa control consisting of fungus inoculated into 990 ul potato dextrosebroth and 10 ul of DMSO (Dimethylsulfoxide). An additional controlconsisting of fungal inoculum in PDB only was performed to ensure thatthe DMSO was not inhibitory to fungal growth. DMSO did not affect any ofthe fungi tested, with the exception of Drechslera portulacae, wheresome inhibition was noted.

Any fungi that showed inhibition were retested. For assays that wererepeated, new stock solutions were made from a second shipment ofpseudomycins. Note that pseudomycin B′ and depsipeptide stock solutionswere remade from first shipment materials because these were notincluded in the second shipment. Several of these fungi showed no growtheven at the lowest levels of pseudomycins added, and were retested athird time using lower levels of pseudomycins (2-0.0625 ug/ml finalconcentration of pseudomycin). Actual concentrations of pseudomycinstested were 2, 1, 0.5, 0.25, 0.125 and 0.0625 ug/ml. A-PO₄ andA-FB=pseudomycin A phosphate salt and free base respectively.

Table 2: Two day results—concentration indicated is lowest level ofcompound (ug /ml) that results is no growth. NI, no inhibition; p(partial), at least 50% inhibited; -, not determined. If number ofassays was greater than one, number of assays performed on the fungus isnoted in parentheses. For several slow-growing fungi, days elapsedbefore observation (if different than two) are noted.

Pseudomycin- A-PO₄ A-FB B B′ C C′ Alternaria helianthi (2x) 25 25 NI 25p at 50 p at 50 Aphanomyces sp. (2x) 50 NI p at 50 25 25 25 Bipolarissorokiniana NI NI NI NI NI NI Botrytis alli (2x) 50 NI NI NI NI NICochizobolus carbonum NI NI NI NI NI NI Diplodia natalensis NI NI NI NINI NI Drechslera portulacae (3x) 0.06 0.25 0.06 0.12 0.06 0.05Drechslera teres NI NI NI NI NI NI Drechslera tritici-repentis (2x) 5025 50 50 50 50 Fusarium avenaceum NI NI NI NI NI NI Fusarium culmorum NINI NI NI NI NI Fusarium oxysporum cubense NI NI NI NI NI NI Fusariumsolani NI p at 50 p at 50 NI p at 50 NI Geotrichim candidum (3x) 12.53.12 1.56 25 3.12 1.56 Monilinia sp. (2x) 12.5 6.25 3.13 25 6.25 3.12Mycosphaeella fijiensis 1 0.5 0.5 0.5 1.56 1.56 (Sigatoka) (7 day, 3x)Mycosphaeella fijiensis 0.12 0.25 0.06 0.5 0.125 0.125 (8088/88) (7 day,3x) Penicillium roqueforti (3x) 0.5 1 0.5 1.56 1.56 1.56 Pestalotiopsismicrospora NE-32 p at 50 p at 50 p at 50 NI NI NI Phoma chrysamthecola —— — — — — Phyllosticta maydis p at 50 p at 50 50 p at 50 p at 50 p at 50Phytophthora cactorum NI NI NI NI NI NI Phytophthora cinnemani NI NI NINI NI NI Phytophthora parasitica NI NI NI NI NI NI Phytophthora ultimumNI NI NI NI NI NI Rhizoctonia solani (2x) 6.25 6.25 6.25 6.25 25 p at 50Sclerotinia sclerotiorum (2x) NI NI p at 50 NI NI 50 Septoria passerinii(3x) 0.125 0.06 0.06 0.5 0.06 0.06 Septoria tritici (3x) 0.25 0.25 0.060.5 0.125 0.125 Stagonospora nodorum NI NI NI NI NI NI Tapesia acuformis(2x) 50 NI NI NI NI NI Tapesia yallundae (2x) 25 12.5 25 6.25 25 6.25Uslilago maydis (3x) 0.5 1 0.5 1.56 0.25 0.25 Verticillium dahliae (2x)p at 50 50 25 NI 25 p at 50

Table 3: Five day results—concentration indicated is lowest level ofcompound (ug /ml) that results in no growth. NI, no inhibition; p(partial), at least 50% inhibited; -, not performed on the fungus isnoted in parentheses. For several slow-growing fungi, days elapsedbefore observation (if different than five) are noted.

Pseudomycin- A-PO₄ A-FB B B′ C C′ Alternaria helianthi (2x) 25 25 50 25NI NI Aphanomnyces sp. (2x) p at 50 NI 50 NI 50 p at 50 Bipolarissorokiniana NI NI NI NI NI NI Botrytis alli (2x) p at 50 NI NI NI NI NICochliobolus carbonum NI NI NI NI NI NI Diplodia natalensis NI NI NI NINI NI Drechslera portulacae (3x, 9 day) 0.06 0.25 0.06 0.25 0.06 0.5Drechslera teres NI NI NI NI NI NI Drechslera tritici-repentis p at 50NI p at 50 p at 50 NI NI Fusarium avenaceum NI NI NI NI NI NI Fusariumculmorum NI NI NI NI NI NI Fusarium oxysporum cubense NI NI NI NI NI NIFusarium solani NI NI NI NI NI NI Geotrichim candidum (3x) 6.25 6.253.12 25 25 6.25 Monilinia sp. (2x) 12.5 12.5 6.25 25 12.5 6.25Mycosphaeella fijiensis (Sigatoka) (3x, 1 1 1 1 1.56 1.56 21 days)Mycosphaeella fijiensis (8088/88) (3x, 0.25 0.25 0.12 1 0.25 0.25 21days) Penicillium roqueforti (3x) 3.12 3.12 6.25 1.56 12.5 p at 50Pestalotiopsis microspora NE-32 NI NI p at 50 NI NI NI Phomachrysamthecola NI p at 50 p at 50 p at 50 NI NI Phyllosticta maydis NINI 50 NI NI NI Phytophthora parasitica NI NI NI NI NI NI Phytophthoracinnemani NI NI NI NI NI NI Phytophthora ultimum NI NI NI NI NI NIPhytophthora cactorum NI NI NI NI NI NI Rhizoctonia solani (2x) 12.5 5050 6.25 p at 50 p at 50 Sclerotinia sclerotiorum (2x) NI NI NI NI NI pat 50 Septoria tritici (3x) 0.25 0.25 0.06 0.5 0.25 0.25 Septoriapasserinii (3x) 0.12 0.06 0.06 0.5 0.06 0.06 Stagonospora nodorum NI NINI NI NI NI Tapesia acuformis (2x) 50 NI NI NI NI NI Tapesia yallundae(2x) 50 25 NI 50 NI NI Ustilago maydis (3x) 1 1 0.5 3.12 0.25 0.25Verticillium dahliae (2x) p at 50 p at 50 p at 50 NI p at 50 p at 50

A review of the above experiments demonstrates that the fingi testedwere best inhibited by one or several of the pseudomycins, rather thanresponding in the same manner to all of the compounds tested. Six fungithat showed no growth even at the lowest concentration of pseudomycinsinitially tested, 1.56 ug/ml. These fungi were retested with even lowerconcentrations of pseudomycins. E.g. Drechslera portulacae appeared tohave no growth even at 0.0625 ug/ml pseudomycin A (PO4), B and C (9days). Two different isolates of Mycosphaeella fijiensis respondeddifferently to the lower doses of pseudomycins. The Sigatoka isolateappeared to be inhibited by each of the pseudomycins down to ˜1 ug/ml.However, 8088/88 isolate was best inhibited by pseudomycin B, with nogrowth at 0.125 ug/ml at 21 days. Septoria tritici and Septoriapasserinii were strongly inhibited by all the pseudomycins, with S.tritici showing no growth at 5 days at 0.0625 ug/ml pseudomycin B, andS. passerinii showing no growth at 5 days at 0.0625 ug/ml pseudomycin A(free base), B, C and C′. Ustilago maydis was best inhibited by eitherpseudomycin C or C′, with no growth at 0.25 ug/ml at 5 days. Some ofthese fungi showed only minor growth inhibition at the highest levels ofpseudomycins tested (e.g. Alternaria helianthi, Aphanomyces sp.,Botrytis alli, Sclerotinia sclerotiorum, Tapesia acuformis, Tapesiayallundae and Verticillium dahliae). Several fungi showed goodinhibition with one or several pseudomycins, but not all. These includeRhizoctonia solani, which was best inhibited by B′ (no growth at 6.25ug/ml at 5 days), Monilinia sp., best inhibited by B and C′ (no growthat 6.25 ug/ml at 5 days), Geotrichim candidum, best inhibited by B (nogrowth at 3.12 ug/ml at 5 days) and Penicillium roqueforti, bestinhibited by B′ (no growth at 1.56 ug/ml at 5 days).

From this data, it can be concluded that the pseudomycins are a group ofselective “natural” fungicides. Some fungi causing infections found inpost harvest crops and other plant species are sensitive to thepseudomycins (e.g. Penicillum and Geotrichum). Pseudomycin showsimpressive activity against M. fijiensis (bananas). Crude preps as wellas purified materials have a potential role in plant disease control.Large scale production of the pseudomycins is feasible and relativelyinexpensive to produce. Natural products are likely to beenvironmentally compatible and potentially safe

REFERENCES

-   1. A. Ballio, F. Bossa, D. DiGiorgio, P. Ferranti, M. Paci, P.    Pucci, A. Scaloni, A. Segre, G. A. Strobel. FEBS Letters, 1994, 355,    96-100.-   2. C. Potera. Science, 1994, 265, 605.-   3. L. Harrision, D. B. Teplow, M. Rinaldi, G. A. Strobel. J. General    Microbiology 1991, 137, 2857-2865.-   4. B. Lam, G. Strobel, L. Harrison, S. Lam. Proc. Natl. Acad. Sci.    1987, 84, 6447-6451.-   5. R. Scheffer, D. Elgersma, G. Strobel. Neth. J. Pl. Path. 1989,    95, 293-304.-   6. R. Scheffer, D. Elgersma, L. A. DeWeger, G. Strobel. Neth. J. Pl.    Path. 1989, 95, 281-292.

1. A method for the protection or treatment of plants and crops against fungal related diseases caused by Mycosphaerella sp. which comprises applying to said plants or crops an effective amount of a pseudomycin composition, wherein the pseudomycin composition comprises a molecule having the chemical formula 1:

wherein R is a lipophilic moiety.
 2. The method according to claim 1, wherein the pseudomycin composition is isolated from Pseudomonas syringae.
 3. The method according to claim 1 wherein said pseudomycin composition comprises an effective amount of molecules selected from the group consisting of Pseudomycin A, Pseudomycin A′, Pseudomycin B, Pseudomycin B′, Pseudomycin C, and Pseudomycin C′.
 4. The method according to claim 1, wherein the pseudomycin composition is applied to said plants or crops as an aqueous suspension, solution or emulsion with a concentration ranging from about 1 to 100 micrograms per ml.
 5. The method according to claim 1, wherein said pseudomycin composition is applied to plants and crops susceptible to Mycosphaerella fijiensis sp.
 6. The method according to claim 1, wherein said plants and crops are selected from the group consisting of bananas, plantains, sunflower, sugar beets, barley, onion, grapes, portulacs, wheat, tomato and corn.
 7. The method according to claim 4, wherein said applying comprises spraying the aqueous suspension, solution or emulsion directly onto said plants and crops.
 8. The method of claim 1, wherein R is a 3,4-dihydroxytetradecanoyl moiety.
 9. The method of claim 1, wherein R is a 3-monohydroxytetradecanoyl moiety.
 10. The method of claim 1, wherein R is a 3,4-dihydroxyhexadecanoyl moiety.
 11. The method of claim 1, wherein R is a 3-monohydroxyhexadecanoyl moiety. 